Abstract Antibiotic-resistant Clostridioides difficile is responsible for more than 29,000 deaths in the US each year and the infection is an urgent threat (as designated by CDC) to public health. The incidence of C. difficile infection (CDI) and disease severity is increasing in recent years due to the emergence of hypervirulent and antibiotic- resistant strains. CDI is caused by the exotoxins TcdA and TcdB secreted by C. difficile in the colon of the host. Current standard treatment with antibiotics is not optimal and is accompanied by a high recurrence rate due to the disruption of host colonization resistance. Moreover, there is no approved prevention against the infection. Our goal is to develop a novel yeast-based immunoprophylactic/therapeutic against both primary and recurrent CDI. We have developed a therapeutic lead by engineering a probiotic yeast, Saccharomyces boulardii (Sb), to secrete an antitoxin ABAB (Sb-ABAB). ABAB is a fusion protein of 4 VHHs targeting 2 distinct neutralizing epitopes in TcdA and 2 in TcdB respectively. ABAB is ultrapotent in neutralizing both TcdA and TcdB and broadly active against toxins from 64 clinical isolates. Our preliminary data showed that oral Sb- ABAB protected mice from both primary and recurrent CDI. The objectives of this Fast-track SBIR are to: 1) phenotypically and genetically characterize Sb-ABAB to ensure its identity and quality; 2) determine pharmacokinetic and safety profiles of Sb-ABAB; 3) develop clinic-compatible formula of Sb-ABAB capsules; and 4) perform IND-enabling efficacy study in a piglet model of CDI. All proposed activities will be guided by an exceptional advisory/consultant team with specialized expertise in business development, biologics regulation, product development and planning, and clinical development. Upon the completion of the proposed studies, we will pursue Phase IIb for GMP manufacturing of Sb-ABAB, GLP toxicology and IND submission in the future.